Allele of human histamine H2 receptor and methods of detection of H2 receptor variants

ABSTRACT

The invention relates to allelic variants of human histamine H 2  receptor genes and primer sequences useful in detecting such genes.

This is a 371 of International Patent Application No. PCT/EP96/00397,with an international filing date of Jan. 30, 1996, now pending, whichclaims priority from United Kingdom Patent Application No. 9503866.7with a filing date of Jan. 30, 1995.

TECHNICAL FIELD

This invention relates to the detection of variations in human histamineH₂ receptors, and more particularly to the development of new compoundsuseful in the sequencing and identification of a human histamine H₂receptor and their use in the diagnosis and treatment of certain humandisorders, for example, brain disorders. The invention also relates tonew compounds and a method for detecting an allelic polymorphicvariation within the human population for the gene encoding thehistamine H₂ receptor and their use in the diagnosis and treatment ofhuman disorders.

BACKGROUND ART

The human H₂ receptor was first identified by Black et al Nature (1972),236, 385-390. This was followed by the demonstration of the receptor inthe mammalian brain by Baudry et al (1975) Nature 253, 362-363, and Haasand Bucher (1975) Nature 255, 634-635. Gantz et al (1991) Biochem.Biophys. Res. Comm. 178,3, 1386-1392 have recently identified thesequence of a human H₂ receptor cDNA from gastric parietal cells byusing the polymerase chain reaction (PCR) and degeneratedoligonucleotide primers whose sequence was obtained from the canine H₂receptor previously cloned by this group, Gantz et al (1991) Proc. Nat.Acad. Sci. USA 88, 429-433. This sequence was characterised as anintronless gene encoding a typical seven transmembrane domain aminergicreceptor protein.

The receptor is coupled to heterotrimeric GTPases (G proteins), butdiffers from other monoamine receptors in this G protein coupledsuperfamily in several respects. The human gastric H₂ receptor isshorter than most other receptors in this class (359 amino acids) andlacks the two serine residues in the fifth transmembrane region (TM5).There exists instead an aspartate and a threonine residue, so far uniquein this region. These two residues may be important for binding with thenitrogen atoms of the imidazole ring of histamine as suggested byBirdsall (1991) Trends in Pharmacological Sci. Jan, 12, 9-10.

Histamine is a natural constituent of many organs and tissues includingthe gastrointestinal tract, the immune system and the brain, Green et al(1987) Agents and Actions 22, 1-15. It is a central neurotransmitter inthe brain and is formed in the posterior hypothalamus from exogenoushistidine by histidine decarboxylase (HDC). It is subsequentlymetabolised by histamine methyltransferase (HMT), Prell et al (1986)Ann. Rev. Neurosci. 9, 209-254. The cell bodies and neuronal pathwaysfor histamine have been mapped in the human brain usingimmunocytochemistry by Panula et al (1990) Neuroscience 34, 127-132. Itscells project from the tuberomamillary nucleus of the posteriorhypothalamus to almost every region of the brain. There are three knownhistamine receptors; H₁, H₂ and H₃, the latter functioning as anautoreceptor. The H₂ receptor specifically has been localised in thehuman brain by Traiffort et al (1992) J. of Neurochem. 59, 1, 290-299.using receptor autoradiography.

Histamine is known to have significant effects in the central nervoussystem (CNS). It has been implicated in the CNS mediated mechanisms ofarousal ever since the sedating effect of H₁ receptor antagonists (eg,.chlorpheniramine, chloropromazine) had been noticed clinically. The useof H₂ receptor antagonists in the human brain however, has shown, thatthese compounds, unlike those acting on the H₁ receptor, do not produceany effect on psychomotor functioning, or a subjective feeling ofsedation or arousal in healthy subjects, White et al (1988)Psychopharmacology 95, 1-14. Some H₂ receptor antagonists (eg.cimetidine) are known to cause confusion in elderly or severelymedically ill patients, perhaps in part due to a co-existinganti-cholinergic effect. H₁ and H₂ receptor antagonists in large doseshave been reported to cause hallucinations, Csillag et al (1973) Med. J.Aust. 1, 653-654, Argawal (1978) J. Am. Med. Assoc. 240, 214. Animalstudies have shown that histamine applied directly to the hippocampus,where there is the highest level of activity of the H₂ receptor, willinduce psychomotor withdrawal and decreased exploratory behaviour. Theabove evidence has led to the conclusion that H₁ receptor systems areexcitatory in the terms of arousal and motivated behaviour whilst H₂receptor systems are inhibitory in this respect, Alvarez and Banzan(1985) Physiol. and Beh. 34, 661-664 and (1986) Physiol. and Beh. 37,39-45, White et al (1988) supra.

The H₂ receptor is a site of action of various compounds used in thetreatment of psychiatric disorders eg. amitriptyline and mianserin,Traiffort et al (1992) supra. Kaminsky et al (1990) The Lancet 335,1351-1352 and (1991) Schizophrenia Bull. 4, 318-319 have reported thesuccessful response of patients with chronic, predominantly negativetype schizophrenia, to the highly specific H₂ receptor antagonistfamotidine. For example in one patient there was a substantialamelioration of the deficit symptoms of schizophrenia (eg apathy, socialwithdrawal, and blunted affect) while on famotidine, relapse in thesesymptoms on withdrawal, and improvement on re-institution of this drug,Kaminski, U.S. Pat. Nos. 5070101 and 5177081. Prell et al (1992)Abstract, part 1, 199.6 Soc. for Neurosci. Annual Meeting, AnaheimCalif. have shown substantially raised levels of N-tele-methylhistamine, a metabolite of histamine in the cerebrospinal fluid ofpatients with schizophrenia which correlates with those patients withthe occurrence of negative symptoms of this disorder assessed using thePsychiatric Symptoms Assessment Scale. These levels were notsignificantly different between patients free from medication and thoseon neuroleptic therapy. It is therefore postulated that there is anincrease in histaminergic activity in patients with chronicschizophrenia.

The disclosures of all the above mentioned publications are incorporatedherein by reference for all purposes.

Additionally, histamine, acting via its receptors, including the H₂receptor, is believed to be critically involved in a number of diseasesof organs other than the brain; these include peptic ulceration,allergic reactions, including asthma, immune-mediated disorders, andpossibly some tumours.

The histamine H₂ receptor is one of many receptors in the body.Compounds used to treat many diseases work by activating a receptor orinhibiting the action if its natural ligand. Variations in receptorsamongst the population are known to be caused by allelic variation andthis variation, can alter the response of a disease to a drug amongstpatients. An example of this would be the response to clozapine, used totreat, schizophrenia associated with allelic variation in the 5-HT_(2A)receptor demonstrated by Arranz el al (1995) Lancet, 346(8970), 281-282.

SUMMARY OF THE INVENTION

The present invention is concerned in one aspect with improvements inthe diagnosis and/or treatment of human neurological and psychiatricdisorders, and more particularly in the diagnosis and treatment ofschizophrenia. In another aspect, the invention is concerned withimprovements in the diagnosis and/or treatment of diseases of othersystems or organs of the human body.

As a first step to the present invention, the Applicants devised a newoligonucleotide probe to the human H₂ receptor mRNA in accordance withthe published cDNA sequence available for the gastric parietal cell.Surprisingly, studies using this probe with in-situ hybridizationhistochemistry on human post-mortem brain tissue produced evidence of amismatch in the nucleotide sequence for the brain H₂ receptor and thesequence for the gastric parietal cell H₂ receptor. This discovery wasmade by recording melt-curve estimations for the optimum hybridizationincubation temperature using the method of Davis et al (1986) "BasicMethods in Molecular Biology" page 77 Elsevier Science Publishing Co. Itwas found that the sequence mismatch is of the order of 10%.

It was apparent, therefore, that there is a hitherto unrecognised alleleor subtype of the human histamine H₂ receptor gene, which may bespecific to the brain.

In a first aspect, the invention provides a sequence for a novel alleleof a human histamine H₂ receptor gene comprising up to six single basesubstitutions compared with the cDNA sequence published by Gantz et al(1991) Biochem Biophys Res Comm 178,3,1386-1392 as follows:

    ______________________________________                                        site of change                                                                             base change                                                                             amino acid alteration                                  ______________________________________                                        398          T - C     Val - Gly                                              525          A - T     Lys - Asn                                              620          A - G     Lys - Asp                                              649          A - G     Asn - Asp                                              692          A - G     Lys - Arg                                              802          G - A     Val - Met                                              ______________________________________                                    

In another aspect, the invention provides a nucleotide sequence codingfor a region of a human histamine H₂ receptor, comprising one or more ofthe following base substitutions compared with the published sequence inGantz et al (1991) supra, and from which the positional notation istaken:

    ______________________________________                                               site of change                                                                         base                                                          ______________________________________                                               398      C                                                                    525      T                                                                    620      G                                                                    649      G                                                                    692      G                                                                    802      A                                                             ______________________________________                                    

The nucleotide sequence of the invention can, for example, comprise thefollowing sequence (as also listed in SEQ ID NO:1):5'CAGCTCGGGTCGCCATCTCTCTGGTCTTAATTTGGGTCATCTCCATTACCCTGTCCTTTCTGTCTATCCACCTGGGGTGGAACAGCAGGAACGAGACCAGCAAGGGCAATCATACCACCTCTAAGTGCAATGTCCAGGTCAATGAAGTGTACGGGCTGGTGGATGGGCTGGTCACCTTCTACCTCCCGCTACTGATCATGTGCATCACCTACTACCGCATCTTCAGGGTCGCCCGGGATCAGGCCAAGAGGATCGATCACATTAGCTCCTGGAAGGCAGCCACCATCAGGGAGCACAGAGCCACAGTGACACTGGCCGCCGTCATGGGGGCCTTCATCATCTGCTGGTTTCCCTACTTCACCGCGTTTGTGTACCGTGGGCTGAGAGGGGATGATGCCATCAATGAGATGTTA3'

As a specific exemplification, the nucleotide sequence of the inventioncan comprise the following sequence (as also listed in SEQ ID NO:2):5'CCAATGGCACAGCCTCTTCCTTTTGCCTGGACTCTACCGCATGCAAGATCACCATCACCGTGGTCCTTGCGGTCCTCATCCTCATCACCGTTGCTGGCAATGTGGTCGTCTGTCTGGCCGTGGGCTTGAACCGCCGGCTCCGCAACCTGACCAATTGTTTCATCGTGTCCTTGGCTATCACTGACCTGCTCCTCGGCCTCCTGGTGCTGCCCTTCTCTGCCATCTACCAGCTGTCCTGCAAGTGGAGCTTTGGCAAGGTCTTCTGCAATATCTACACCAGCCTGGATGTGATGCTCTGCACAGCCTCCATTCTTAACCTCTTCATGATCAGCCTCGACCGGTACTGCGCTGTCATGGACCCACTGCGGTACCCTGTGCTGGTCACCCCAGCTCGGGTCGCCATCTCTCTGGTCTTAATTTGGGTCATCTCCATTACCCTGTCCTTTCTGTCTATCCACCTGGGGTGGAACAGCAGGAACGAGACCAGCAAGGGCAATCATACCACCTCTAAGTGCAATGTCCAGGTCAATGAAGTGTACGGGCTGGTGGATGGGCTGGTCACCTTCTACCTCCCGCTACTGATCATGTGCATCACCTACTACCGCATCTTCAGGGTCGCCCGGGATCAGGCCAAGAGGATCGATCACATTAGCTCCTGGAAGGCAGCCACCATCAGGGAGCACAGAGCCACAGTGACACTGGCCGCCGTCATGGGGGCCTTCATCATCTGCTGGTTTCCCTACTTCACCGCGTTTGTGTACCGTGGGCTGAGAGGGGATGATGCCATCAATGAGATGTTAGAAGCCATCGTTCTGTGGCTGGGCTATGCCAACTCAGCCCTGAACCCCATCCTGTATGCTGCGCTGAACAGAGACTTCCGCACCGGGTACCAACAGCTCTTCTGCTGCAGGCTGGCCAACCGCAACTCCCACAAAACTTCTCTGAGGTCCAACGCCTCTCAGCTGTCCAGGACCCAAAGCCGAGAACCCAGGCAACAGGAAGAGAAACCCCTGAAGCTCCAGGTGTGGAGTGGGACAGAAGTCACG3'

In another aspect of the invention, a series of new oligonucleotideprimers have been developed for the identification of sequences in asample comprising a human histamine H₂ receptor DNA, cDNA or RNAoriginating from a tissue sample or body fluid.

In this aspect, the invention provides new oligonucleotides, suitablefor use as primers for the amplification of DNA corresponding to aregion of a human histamine H₂ receptor, having nucleotide sequencesselected from:

1) 5' CCAATGGCACAGCCTCTT 3' (as listed in SEQ ID NO: 3)

2) 5' CGTGACTTCTGTCCCACT 3' (as listed in SEQ ID NO: 4)

3) 5' CCAGGCAACAGGAAGAGA 3' (as listed in SEQ ID NO: 5)

4) 5' TCTCTTCCTGTTGCCTGG 3' (as listed in SEQ ID NO: 6)

5) 5' GCAGCAGAAGAGCTGTTG 3' (as listed in SEQ ID NO: 7)

6) 5' TCCAGGTCAATGAAGTGT 3' (as listed in SEQ ID NO: 8)

7) 5' ACACTTCATTGACCTGGA 3' (as listed in SEQ ID NO: 9)

8) 5' CCAAGAGGATCAATCACA 3' (as listed in SEQ ID NO: 10)

9) 5' TGTGATTGATCCTCTTGG 3' (as listed in SEQ ID NO: 11)

and a diagnostic kit comprising one or more of the new oligonucleotides.

The direction and base start numbers for the novel oligonucleotideprimers are as follows:

    ______________________________________                                        Primer            Base Start No.                                              ______________________________________                                        1)      Upstream      8                                                       2)      Downstream    1036 and 1095                                           3)      Upstream      995                                                     4)      Downstream    1012 (no. 3) in reverse)                                5)      Downstream    898 and 1171                                            6)      Upstream      527                                                     7)      Downstream    544 (no. 6) in reverse)                                 8)      Upstream      638                                                     9)      Downstream    655 (no. 8) in reverse)                                 ______________________________________                                    

Information on the human histamine H₂ receptor was obtained from the MRCDaresbury database accessing "Genem 61" File no. M64799--Human histamineH₂ receptor gene.

The above mentioned substitutions alter and in some instances introduceor remove new sites for cleavage by specific restriction endonucleasesas follows:

    ______________________________________                                        base change site                                                                           alters restriction map of:                                       ______________________________________                                        398          AluI, AvaI, BspWI, BsrI, CviJI                                   525                                                                           620          Eco57                                                            649          ClaI, Sau3A, TaqI                                                692                                                                           802          MnlI                                                             ______________________________________                                    

The invention further provides a diagnostic kit comprising one or moreof the new oligonucleotide primers and, preferably, one or more of theabove mentioned endonucleases, optionally with one or more buffers.

A kit may be used to establish genotype or base variations. Thisinformation may be used in predicting an individuals diseasesusceptibility, disease course, prognosis and/or response to treatmentas would be understood by those skilled in the art from the disclosurecontained herein. The treatment response or efficacy which may bepredicted may include drug treatment such as for example, use of H₂receptor antagonist like famotidine or other forms of treatment such associal or psychological intervention.

Eucaryotic expression vectors comprising a DNA sequence coding for aprotein and or a peptide according to the invention are new materialsand are also included in the invention. Host cells, for example, clonedhuman cell lines, such as NTera 2 c.d1, can be transformed using the newexpression vectors and are also included in the invention.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a photograph of an agarose gel containing isolated nucleicacids of the invention.

DETAILED DESCRIPTION

Expression vectors and host cells transformed thereby, in accordancewith the invention, can be prepared, for example, as detailed below, andthe encoded protein studied, by one or more of the following exemplarymethods:

1. Total RNA is extracted from homogenised human tissue, eg. brain, bythe acid guanidine thiocyanate method (Chomczynski & Saach (1987), anal.Biochem. 161, 156-159). Messenger RNA (mRNA) is purified from this byhybridisation of oligo(d)T to the polyadenylated tails present on themajority of mRNA's, for example, using the Promega PolyAttract® system.Reverse transcription of the mRNA using specific reverse transcriptaseenzyme, eg. Superscipt II, Gibco BRL, is followed by PCR amplificationof the resultant product using specific oligonucleotide primers, forexample, those previously described. The resulting amplified cDNA isligated into an expression vector, eg. pGEMEX®-1 vector available fromPromega. Competent cells, eg. bacterial strain JM109(DE3), alsoavailable from Promega, are transformed using this vector, effectivetransforms selected and cultured. Expression of the encoded protein isthen induced with a suitable promotor, eg. IPTG, and the expressedprotein purified from the cell culture using standard biochemicalprocedures, eg. cell lysis and polyacrylamide gel electrophoresis.

2. An alternative method for examining the functional protein encoded bythe cDNA described above, is to induce transcription of the cloned cDNA,as above, and to purify the specific mRNA from the cell culture asdescribed. The purified mRNA is introduced into competent cells, eg.frog oocytes or Chinese hamster ovary cells, and the function of theencoded protein studied by standard pharmacological and physiologicaltechniques, eg. microelectrode recording and receptor bindingtechniques.

3. As 1 above, but introducing the cDNA into a coupledtranscription-translation system, eg. TNT, Promega with subsequentpurification and analysis of the encoded protein as described.

The invention is illustrated by the following Examples:

EXAMPLE 1

This example describes the identification and sequencing of an allelichuman H₂ receptor gene using certain novel oligonucleotide primersaccording to the invention.

A polymerase chain reaction (PCR) product is prepared from human DNA.

DNA was extracted from human brain tissue by first pulverizingapproximately 1 g of tissue in liquid nitrogen then adding to 10 mllysis buffer (0.32M sucrose, 10 mM Tris, 5 mM magnesium chloride 1%Triton X-100 pH8.0). This solution was centrifuged (9,000 rpm 15 mins)to pellet the tissue, the lysis buffer was drawn off and the pelletresuspended in 4.5 ml 75 mM sodium chloride, 24 mM EDTA. This solutionwas then incubated for 3 hours with 250 μl 10% SDS and 2 mg proteinase Kat 56° C. This aqueous phase was then extracted twice with 5 ml ofphenol:chloroform:isoamyl alcohol (25:24:1). Then sodium acetate to 0.3MpH7.5 and 2 volumes of ethanol (at -20° C.) were added to the aqueousphase and the DNA hooked out into TE buffer. The concentration of theDNA was determined by measuring the optical density of the sample, at awavelength of 260 nm.

The DNA was then amplified by the polymerase chain reaction using theoligonucleotide primers 1) and 2) (as hereinbefore described) for 36cycles. The timing for each cycle was as follows; 1 min at 94° C., 1.5min at 56° C. and 2 mins at 72° C., this was then followed by a 10 minextension at 72° C. (Amplitaq DNA polymerase Perkin-Elmer Cetus). Thisreaction produced a DNA fragment of 1047 base pair when analyzed by gelelectrophoresis.

Following PCR amplification of the DNA, the PCR products wereimmediately ligated and cloned into the TA cloning system (InvitroGen).The transformed cells were plated onto Luria-Bertani plates containing50 μl/ml amplicillin and 1.6 mg X-Gal. Plates were then incubatedovernight at 37° C., then moved to 4° C. for 4 hours to allow for colourdevelopment. Positive (white colonies) were then analyzed by growing a 5ml culture overnight at 37° C. extracting the plasmids (Qiaspinminipreps (Qiagen)) and performing an EcoRI digest to ensure the correctsize product was contained in the plasmid. The plasmid used to clone thePCR product is the pCR™II Vector, which is transformed into One Shot™INVαF' Competent cells.

Both strands of the cloned PCR product were sequenced using thedideoxynucleotide chain-terminated method, carried out with Sequenaseversion 2.0 (Amersham/USB). Partial sequencing of short stretches of thecloned DNA utilised all the oligonucleotide primers 1) to 9)hereinbefore described. The cloned PCR product was shown to be identicalto the gastric cDNA of Gantz et al except for the previously mentionedsix single base changes.

Results and discussions

The sequence derived from the above described method is listed below andin SEQ ID NO:2.5'CCAATGGCACAGCCTCTTCCTTTTGCCTGGACTCTACCGCATGCAAGATCACCATCACCGTGGTCCTTGCGGTCCTCATCCTCATCACCGTTGCTGGCAATGTGGTCGTCTGTCTGGCCGTGGGCTTGAACCGCCGGCTCCGCAACCTGACCAATTGTTTCATCGTGTCCTTGGCTATCACTGACCTGCTCCTCGGCCTCCTGGTGCTGCCCTTCTCTGCCATCTACCAGCTGTCCTGCAAGTGGAGCTTTGGCAAGGTCTTCTGCAATATCTACACCAGCCTGGATGTGATGCTCTGCACAGCCTCCATTCTTAACCTCTTCATGATCAGCCTCGACCGGTACTGCGCTGTCATGGACCCACTGCGGTACCCTGTGCTGGTCACCCCAGCTCGGGTCGCCATCTCTCTGGTCTTAATTTGGGTCATCTCCATTACCCTGTCCTTTCTGTCTATCCACCTGGGGTGGAACAGCAGGAACGAGACCAGCAAGGGCAATCATACCACCTCTAAGTGCAATGTCCAGGTCAATGAAGTGTACGGGCTGGTGGATGGGCTGGTCACCTTCTACCTCCCGCTACTGATCATGTGCATCACCTACTACCGCATCTTCAGGGTCGCCCGGGATCACGCCAAGAGGATCGATCACATTAGCTCCTGGAAGGCAGCCACCATCAGGGAGCACAGAGCCACAGTGACACTGGCCGCCGTCATGGGGGCCTTCATCATCTGCTGGTTTCCCTACTTCACCGCGTTTGTGTACCGTGGGCTGAGAGGGGATGATGCCATCAATGAGATGTTAGAAGCCATCGTTCTGTGGCTGGGCTATGCCAACTCAGCCCTGAACCCCATCCTGTATGCTGCGCTGAACAGAGACTTCCGCACCGGGTACCAACAGCTCTTCTGCTGCAGGCTGGCCAACCGCAACTCCCACAAAACTTCTCTGAGGTCCAACGCCTCTCAGCTGTCCAGGACCCAAAGCCGAGAACCCAGGCAACAGGAAGAGAAACCCCTGAAGCTCCAGGTGTGGAGTGGGACAGAAGTCACG3'

EXAMPLE 2

This example describes the confirmation of the presence of the basechanges in a larger population. This is made possible by an assay basedupon PCR amplification of a 909 base pair fragment of the H₂ receptorgene from human DNA, followed by cleavage utilising specific restrictionendonucleases. It will apparent to those skilled in the art that singlebase changes could be detected using other techniques known to those inthe art which include single stranded confirmational polymorphisms(sscp), chemical cleavage, PCR thermoligase reactions etc.

Samples of blood are collected from human volunteers into EDTA coatedtube, 1 ml of this blood is heated to 100° C. for 15 minutes then spunin a microcentrifuge at 13,000Xg for 15 minutes. This supernatant iscollected, and the cell debris is discarded. Then 0.5-3 μl of thissupernatant is utilised as template DNA for a PCR reaction to amplify aportion of the receptor gene between bases 8 and 915. The conditions forthis PCR reaction are 3 mM MgCl₂ (Gibco BRL),1X PCR buffer (Gibco BRL) 1mM of each dATP, dGTP, dGTP and dTTP (Promega) 10 pmoles of each ofoligonucleotide primers 1) and 5) (hereinbefore described) and 1 unitTaq DNA polymerase (Gibco BRL), in a total volume adjusted to 50 μl bysterile DNAse free water. This mix is then subjected to the followingconditions; 96° C. 5 minutes, then 35 cycles of 96° C. for 1 minute, 56°C. for 1 minute, 72° C. for 1 minute and 20 seconds.

10 μl of the resultant products are then analysed on a 1% agarose gel toensure that the above reaction is correctly amplifying the target DNAfragment. Then 11.5 μl of the PCR mix is added to 2 units Taq1restriction endonuclease (Fermentas) and 1.5 μl of 10X buffer andincubated at 65° C. for 3-24 hours. The products of this reaction arethen analysed on a 2.5% agarose gel. If the original sequence describedby Gantz (nominated H₂ A) has been amplified, then bands of 574 and 335base pairs are seen which indicates that the individual is an A/Ahomozygote. If the sequence described in Example 1 (nominated H₂ B) hasbeen amplified, then following the TaqI cleavage of the PCR product,bands of 335, 306 and 268 base pairs can be seen, indicating that thatindividual is a B/B homozygote. If bands of 574, 335, 306 and 268 basepairs can be seen, then that individual is an A/B heterozygote.

Typical results are illustrated in FIG. 1, which shows a 2.5% TBEAgarose gel stained with ethidium bromide, showing TαqI digestionpatterns of a 909 base pair PCR fragment, from 4 separate individuals.

Lanes A+G - 100 base pair DNA marker (Gibco BRL)

Lanes B+F - Band pattern indicative of an A/B heterozygote

Lanes C+E - Band pattern indicative of an B/B homozygote

Lane D - Blank

Arrows indicated the sizes of the DNA fragments in lanes B to F.

Primers: Oswell DNA Services)

1) upstream: 5' CCAATGGCACAGCCTCTT 3' (as in SEQ ID NO:1)

2) upstream: 5' CCAGGCAACAGGAAGAGA 3' (as in SEQ ID NO:5)

5) downstream: 5' GCAGCAGAAGAGCTGTTG 3' (as in SEQ ID NO:7)

EXAMPLE 3

A method as described in example 2 is applied to a series of DNA samplesextracted from schizophrenic individuals, their first degree relatives,and normal controls. There is observed a statistically significantdifference of P less than 0.01 the pattern seen in the genotype of theseindividuals, as described in the table below:

    ______________________________________                                                  H.sub.2  Genotype                                                   Diagnosis   A/A         A/B     B/B                                           ______________________________________                                        Controls    12.1%       48.5%   39.4%                                         Schizophrenia                                                                              9.8%       26.8%   63.4%                                         1st degree   6.1%       12.1%   81.8%                                         relatives                                                                     ______________________________________                                    

Discussion

The variable sequence is explained by a polymorphic allelic variationwithin the human population for the gene encoding the H₂ receptorprotein. This allelic polymorphism may lead to substantial variation inthe effect of activation of the encoded receptor by histamine, either inthe efficacy of histamine binding, the duration of activation, or theintracellular effects of such activation. It is envisaged that suchvariation resulting from allelic polymorphism may underlinesusceptibility to specific disorders, both affecting the brain and/orinvolving other systems or organs. In summary, this variation in thehuman H₂ receptor gene and its products, including, for example, mRNAand proteins, could be used as a method of establishing individual riskto a particular psychiatric or neurological or other illness eg.schizophrenia.

Alternative embodiments of the invention can be envisaged by thoseskilled in the art from the information contained herein. All suchalternative embodiments are intended to lie within the scope of thisapplication.

The reader's attention is directed to all papers and documents which arefiled concurrently with this specification and which are open to publicinspection with this specification, and the contents of all such papersand documents are incorporated herein by reference.

All the features disclosed in this specification (including anyaccompanying claims, abstract and drawings), and/or all of the steps orany method or process so disclosed, may be combined in any combination,except combinations where at least some of such features and/or stepsare mutually exclusive.

Each feature disclosed in this specification (including any accompanyingclaims, abstract and drawings), may be replaced by alternative featuresserving the same, equivalent or similar purpose, unless expressly statedotherwise. Thus, unless expressly stated otherwise, each featuredisclosed is one example only of a generic series of equivalent orsimilar features.

The invention is not restricted to the details of the foregoingembodiments. This invention extends to any novel one, or any novelcombination, of the features disclosed in this specification (includingany accompanying claims, abstract and drawings), or to any novel one, orany novel combination, of the steps of any method or process sodisclosed.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - <160> NUMBER OF SEQ ID NOS: 15                                              - <210> SEQ ID NO 1                                                           <211> LENGTH: 413                                                             <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: CDS                                                           <222> LOCATION: (3)...(413)                                                   - <400> SEQUENCE: 1                                                           - ca gct cgg gtc gcc atc tct ctg gtc tta att - # tgg gtc atc tcc att            47                                                                          #Ile Trp Val Ile Ser Ileer Leu Val Leu                                        #  15                                                                         - acc ctg tcc ttt ctg tct atc cac ctg ggg tg - #g aac agc agg aac gag           95                                                                          Thr Leu Ser Phe Leu Ser Ile His Leu Gly Tr - #p Asn Ser Arg Asn Glu           #                 30                                                          - acc agc aag ggc aat cat acc acc tct aag tg - #c aat gtc cag gtc aat          143                                                                          Thr Ser Lys Gly Asn His Thr Thr Ser Lys Cy - #s Asn Val Gln Val Asn           #             45                                                              - gaa gtg tac ggg ctg gtg gat ggg ctg gtc ac - #c ttc tac ctc ccg cta          191                                                                          Glu Val Tyr Gly Leu Val Asp Gly Leu Val Th - #r Phe Tyr Leu Pro Leu           #         60                                                                  - ctg atc atg tgc atc acc tac tac cgc atc tt - #c agg gtc gcc cgg gat          239                                                                          Leu Ile Met Cys Ile Thr Tyr Tyr Arg Ile Ph - #e Arg Val Ala Arg Asp           #     75                                                                      - cag gcc aag agg atc gat cac att agc tcc tg - #g aag gca gcc acc atc          287                                                                          Gln Ala Lys Arg Ile Asp His Ile Ser Ser Tr - #p Lys Ala Ala Thr Ile           # 95                                                                          - agg gag cac aga gcc aca gtg aca ctg gcc gc - #c gtc atg ggg gcc ttc          335                                                                          Arg Glu His Arg Ala Thr Val Thr Leu Ala Al - #a Val Met Gly Ala Phe           #               110                                                           - atc atc tgc tgg ttt ccc tac ttc acc gcg tt - #t gtg tac cgt ggg ctg          383                                                                          Ile Ile Cys Trp Phe Pro Tyr Phe Thr Ala Ph - #e Val Tyr Arg Gly Leu           #           125                                                               #          413     cc atc aat gag atg tta                                     Arg Gly Asp Asp Ala Ile Asn Glu Met Leu                                       #       135                                                                   - <210> SEQ ID NO 2                                                           <211> LENGTH: 1046                                                            <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: CDS                                                           <222> LOCATION: (3)...(1046)                                                  - <400> SEQUENCE: 2                                                           - cc aat ggc aca gcc tct tcc ttt tgc ctg gac - # tct acc gca tgc aag            47                                                                          #Asp Ser Thr Ala Cys Lyser Phe Cys Leu                                        #  15                                                                         - atc acc atc acc gtg gtc ctt gcg gtc ctc at - #c ctc atc acc gtt gct           95                                                                          Ile Thr Ile Thr Val Val Leu Ala Val Leu Il - #e Leu Ile Thr Val Ala           #                 30                                                          - ggc aat gtg gtc gtc tgt ctg gcc gtg ggc tt - #g aac cgc cgg ctc cgc          143                                                                          Gly Asn Val Val Val Cys Leu Ala Val Gly Le - #u Asn Arg Arg Leu Arg           #             45                                                              - aac ctg acc aat tgt ttc atc gtg tcc ttg gc - #t atc act gac ctg ctc          191                                                                          Asn Leu Thr Asn Cys Phe Ile Val Ser Leu Al - #a Ile Thr Asp Leu Leu           #         60                                                                  - ctc ggc ctc ctg gtg ctg ccc ttc tct gcc at - #c tac cag ctg tcc tgc          239                                                                          Leu Gly Leu Leu Val Leu Pro Phe Ser Ala Il - #e Tyr Gln Leu Ser Cys           #     75                                                                      - aag tgg agc ttt ggc aag gtc ttc tgc aat at - #c tac acc agc ctg gat          287                                                                          Lys Trp Ser Phe Gly Lys Val Phe Cys Asn Il - #e Tyr Thr Ser Leu Asp           # 95                                                                          - gtg atg ctc tgc aca gcc tcc att ctt aac ct - #c ttc atg atc agc ctc          335                                                                          Val Met Leu Cys Thr Ala Ser Ile Leu Asn Le - #u Phe Met Ile Ser Leu           #               110                                                           - gac cgg tac tgc gct gtc atg gac cca ctg cg - #g tac cct gtg ctg gtc          383                                                                          Asp Arg Tyr Cys Ala Val Met Asp Pro Leu Ar - #g Tyr Pro Val Leu Val           #           125                                                               - acc cca gct cgg gtc gcc atc tct ctg gtc tt - #a att tgg gtc atc tcc          431                                                                          Thr Pro Ala Arg Val Ala Ile Ser Leu Val Le - #u Ile Trp Val Ile Ser           #       140                                                                   - att acc ctg tcc ttt ctg tct atc cac ctg gg - #g tgg aac agc agg aac          479                                                                          Ile Thr Leu Ser Phe Leu Ser Ile His Leu Gl - #y Trp Asn Ser Arg Asn           #   155                                                                       - gag acc agc aag ggc aat cat acc acc tct aa - #g tgc aat gtc cag gtc          527                                                                          Glu Thr Ser Lys Gly Asn His Thr Thr Ser Ly - #s Cys Asn Val Gln Val           160                 1 - #65                 1 - #70                 1 -       #75                                                                           - aat gaa gtg tac ggg ctg gtg gat ggg ctg gt - #c acc ttc tac ctc ccg          575                                                                          Asn Glu Val Tyr Gly Leu Val Asp Gly Leu Va - #l Thr Phe Tyr Leu Pro           #               190                                                           - cta ctg atc atg tgc atc acc tac tac cgc at - #c ttc agg gtc gcc cgg          623                                                                          Leu Leu Ile Met Cys Ile Thr Tyr Tyr Arg Il - #e Phe Arg Val Ala Arg           #           205                                                               - gat cag gcc aag agg atc gat cac att agc tc - #c tgg aag gca gcc acc          671                                                                          Asp Gln Ala Lys Arg Ile Asp His Ile Ser Se - #r Trp Lys Ala Ala Thr           #       220                                                                   - atc agg gag cac aga gcc aca gtg aca ctg gc - #c gcc gtc atg ggg gcc          719                                                                          Ile Arg Glu His Arg Ala Thr Val Thr Leu Al - #a Ala Val Met Gly Ala           #   235                                                                       - ttc atc atc tgc tgg ttt ccc tac ttc acc gc - #g ttt gtg tac cgt ggg          767                                                                          Phe Ile Ile Cys Trp Phe Pro Tyr Phe Thr Al - #a Phe Val Tyr Arg Gly           240                 2 - #45                 2 - #50                 2 -       #55                                                                           - ctg aga ggg gat gat gcc atc aat gag atg tt - #a gaa gcc atc gtt ctg          815                                                                          Leu Arg Gly Asp Asp Ala Ile Asn Glu Met Le - #u Glu Ala Ile Val Leu           #               270                                                           - tgg ctg ggc tat gcc aac tca gcc ctg aac cc - #c atc ctg tat gct gcg          863                                                                          Trp Leu Gly Tyr Ala Asn Ser Ala Leu Asn Pr - #o Ile Leu Tyr Ala Ala           #           285                                                               - ctg aac aga gac ttc cgc acc ggg tac caa ca - #g ctc ttc tgc tgc agg          911                                                                          Leu Asn Arg Asp Phe Arg Thr Gly Tyr Gln Gl - #n Leu Phe Cys Cys Arg           #       300                                                                   - ctg gcc aac cgc aac tcc cac aaa act tct ct - #g agg tcc aac gcc tct          959                                                                          Leu Ala Asn Arg Asn Ser His Lys Thr Ser Le - #u Arg Ser Asn Ala Ser           #   315                                                                       - cag ctg tcc agg acc caa agc cga gaa ccc ag - #g caa cag gaa gag aaa         1007                                                                          Gln Leu Ser Arg Thr Gln Ser Arg Glu Pro Ar - #g Gln Gln Glu Glu Lys           320                 3 - #25                 3 - #30                 3 -       #35                                                                           #   1046g aag ctc cag gtg tgg agt ggg aca ga - #a gtc acg                     Pro Leu Lys Leu Gln Val Trp Ser Gly Thr Gl - #u Val Thr                       #               345                                                           - <210> SEQ ID NO 3                                                           <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial sequence                                           <220> FEATURE:                                                                <221> NAME/KEY: Synthetic Oligonucleotide                                     - <400> SEQUENCE: 3                                                           #  18              tt                                                         - <210> SEQ ID NO 4                                                           <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial sequence                                           <220> FEATURE:                                                                <221> NAME/KEY: Synthetic Oligonucleotide                                     - <400> SEQUENCE: 4                                                           #  18              ct                                                         - <210> SEQ ID NO 5                                                           <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial sequence                                           <220> FEATURE:                                                                <221> NAME/KEY: Synthetic Oligonucleotide                                     - <400> SEQUENCE: 5                                                           #  18              ga                                                         - <210> SEQ ID NO 6                                                           <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial sequence                                           <220> FEATURE:                                                                <221> NAME/KEY: Synthetic Oligonucleotide                                     - <400> SEQUENCE: 6                                                           #  18              gg                                                         - <210> SEQ ID NO 7                                                           <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial sequence                                           <220> FEATURE:                                                                <221> NAME/KEY: Synthetic Oligonucleotide                                     - <400> SEQUENCE: 7                                                           #  18              tg                                                         - <210> SEQ ID NO 8                                                           <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial sequence                                           <220> FEATURE:                                                                <221> NAME/KEY: Synthetic Oligonucleotide                                     - <400> SEQUENCE: 8                                                           #  18              gt                                                         - <210> SEQ ID NO 9                                                           <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial sequence                                           <220> FEATURE:                                                                <221> NAME/KEY: Synthetic Oligonucleotide                                     - <400> SEQUENCE: 9                                                           #  18              ga                                                         - <210> SEQ ID NO 10                                                          <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial sequence                                           <220> FEATURE:                                                                <221> NAME/KEY: Synthetic Oligonucleotide                                     - <400> SEQUENCE: 10                                                          #  18              ca                                                         - <210> SEQ ID NO 11                                                          <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial sequence                                           <220> FEATURE:                                                                <221> NAME/KEY: Synthetic Oligonucleotide                                     - <400> SEQUENCE: 11                                                          #  18              gg                                                         - <210> SEQ ID NO 12                                                          <211> LENGTH: 137                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 12                                                          - Ala Arg Val Ala Ile Ser Leu Val Leu Ile Tr - #p Val Ile Ser Ile Thr         #                 15                                                          - Leu Ser Phe Leu Ser Ile His Leu Gly Trp As - #n Ser Arg Asn Glu Thr         #             30                                                              - Ser Lys Gly Asn His Thr Thr Ser Lys Cys As - #n Val Gln Val Asn Glu         #         45                                                                  - Val Tyr Gly Leu Val Asp Gly Leu Val Thr Ph - #e Tyr Leu Pro Leu Leu         #     60                                                                      - Ile Met Cys Ile Thr Tyr Tyr Arg Ile Phe Ar - #g Val Ala Arg Asp Gln         # 80                                                                          - Ala Lys Arg Ile Asp His Ile Ser Ser Trp Ly - #s Ala Ala Thr Ile Arg         #                 95                                                          - Glu His Arg Ala Thr Val Thr Leu Ala Ala Va - #l Met Gly Ala Phe Ile         #           110                                                               - Ile Cys Trp Phe Pro Tyr Phe Thr Ala Phe Va - #l Tyr Arg Gly Leu Arg         #       125                                                                   - Gly Asp Asp Ala Ile Asn Glu Met Leu                                         #   135                                                                       - <210> SEQ ID NO 13                                                          <211> LENGTH: 348                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 13                                                          - Asn Gly Thr Ala Ser Ser Phe Cys Leu Asp Se - #r Thr Ala Cys Lys Ile         #                 15                                                          - Thr Ile Thr Val Val Leu Ala Val Leu Ile Le - #u Ile Thr Val Ala Gly         #             30                                                              - Asn Val Val Val Cys Leu Ala Val Gly Leu As - #n Arg Arg Leu Arg Asn         #         45                                                                  - Leu Thr Asn Cys Phe Ile Val Ser Leu Ala Il - #e Thr Asp Leu Leu Leu         #     60                                                                      - Gly Leu Leu Val Leu Pro Phe Ser Ala Ile Ty - #r Gln Leu Ser Cys Lys         # 80                                                                          - Trp Ser Phe Gly Lys Val Phe Cys Asn Ile Ty - #r Thr Ser Leu Asp Val         #                 95                                                          - Met Leu Cys Thr Ala Ser Ile Leu Asn Leu Ph - #e Met Ile Ser Leu Asp         #           110                                                               - Arg Tyr Cys Ala Val Met Asp Pro Leu Arg Ty - #r Pro Val Leu Val Thr         #       125                                                                   - Pro Ala Arg Val Ala Ile Ser Leu Val Leu Il - #e Trp Val Ile Ser Ile         #   140                                                                       - Thr Leu Ser Phe Leu Ser Ile His Leu Gly Tr - #p Asn Ser Arg Asn Glu         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Thr Ser Lys Gly Asn His Thr Thr Ser Lys Cy - #s Asn Val Gln Val Asn         #               175                                                           - Glu Val Tyr Gly Leu Val Asp Gly Leu Val Th - #r Phe Tyr Leu Pro Leu         #           190                                                               - Leu Ile Met Cys Ile Thr Tyr Tyr Arg Ile Ph - #e Arg Val Ala Arg Asp         #       205                                                                   - Gln Ala Lys Arg Ile Asp His Ile Ser Ser Tr - #p Lys Ala Ala Thr Ile         #   220                                                                       - Arg Glu His Arg Ala Thr Val Thr Leu Ala Al - #a Val Met Gly Ala Phe         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Ile Ile Cys Trp Phe Pro Tyr Phe Thr Ala Ph - #e Val Tyr Arg Gly Leu         #               255                                                           - Arg Gly Asp Asp Ala Ile Asn Glu Met Leu Gl - #u Ala Ile Val Leu Trp         #           270                                                               - Leu Gly Tyr Ala Asn Ser Ala Leu Asn Pro Il - #e Leu Tyr Ala Ala Leu         #       285                                                                   - Asn Arg Asp Phe Arg Thr Gly Tyr Gln Gln Le - #u Phe Cys Cys Arg Leu         #   300                                                                       - Ala Asn Arg Asn Ser His Lys Thr Ser Leu Ar - #g Ser Asn Ala Ser Gln         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Leu Ser Arg Thr Gln Ser Arg Glu Pro Arg Gl - #n Gln Glu Glu Lys Pro         #               335                                                           - Leu Lys Leu Gln Val Trp Ser Gly Thr Glu Va - #l Thr                         #           345                                                               - <210> SEQ ID NO 14                                                          <211> LENGTH: 1080                                                            <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: CDS                                                           <222> LOCATION: (1)...(1077)                                                  - <400> SEQUENCE: 14                                                          - atg gca ccc aat ggc aca gcc tct tcc ttt tg - #c ctg gac tct acc gca           48                                                                          Met Ala Pro Asn Gly Thr Ala Ser Ser Phe Cy - #s Leu Asp Ser Thr Ala           #                 15                                                          - tgc aag atc acc atc acc gtg gtc ctt gcg gt - #c ctc atc ctc atc acc           96                                                                          Cys Lys Ile Thr Ile Thr Val Val Leu Ala Va - #l Leu Ile Leu Ile Thr           #             30                                                              - gtt gct ggc aat gtg gtc gtc tgt ctg gcc gt - #g ggc ttg aac cgc cgg          144                                                                          Val Ala Gly Asn Val Val Val Cys Leu Ala Va - #l Gly Leu Asn Arg Arg           #         45                                                                  - ctc cgc aac ctg acc aat tgt ttc atc gtg tc - #c ttg gct atc act gac          192                                                                          Leu Arg Asn Leu Thr Asn Cys Phe Ile Val Se - #r Leu Ala Ile Thr Asp           #     60                                                                      - ctg ctc ctc ggc ctc ctg gtg ctg ccc ttc tc - #t gcc atc tac cag ctg          240                                                                          Leu Leu Leu Gly Leu Leu Val Leu Pro Phe Se - #r Ala Ile Tyr Gln Leu           # 80                                                                          - tcc tgc aag tgg agc ttt ggc aag gtc ttc tg - #c aat atc tac acc agc          288                                                                          Ser Cys Lys Trp Ser Phe Gly Lys Val Phe Cy - #s Asn Ile Tyr Thr Ser           #                 95                                                          - ctg gat gtg atg ctc tgc aca gcc tcc att ct - #t aac ctc ttc atg atc          336                                                                          Leu Asp Val Met Leu Cys Thr Ala Ser Ile Le - #u Asn Leu Phe Met Ile           #           110                                                               - agc ctc gac cgg tac tgc gct gtc atg gac cc - #a ctg cgg tac cct gtg          384                                                                          Ser Leu Asp Arg Tyr Cys Ala Val Met Asp Pr - #o Leu Arg Tyr Pro Val           #       125                                                                   - ctg gtc acc cca gtt cgg gtc gcc atc tct ct - #g gtc ata att tgg gtc          432                                                                          Leu Val Thr Pro Val Arg Val Ala Ile Ser Le - #u Val Ile Ile Trp Val           #   140                                                                       - atc tcc att acc ctg tcc ttt ctg tct atc ca - #c ctg ggg tgg aac agc          480                                                                          Ile Ser Ile Thr Leu Ser Phe Leu Ser Ile Hi - #s Leu Gly Trp Asn Ser           145                 1 - #50                 1 - #55                 1 -       #60                                                                           - agg aac gag acc agc aag ggc aat cat acc ac - #c tct aag tgc aaa gtc          528                                                                          Arg Asn Glu Thr Ser Lys Gly Asn His Thr Th - #r Ser Lys Cys Lys Val           #               175                                                           - cag gtc aat gaa gtg tac ggg ctg gtg gat gg - #g ctg gtc acc ttc tac          576                                                                          Gln Val Asn Glu Val Tyr Gly Leu Val Asp Gl - #y Leu Val Thr Phe Tyr           #           190                                                               - ctc ccg cta ctg atc atg tgc atc acc tac ta - #c cgc atc ttc aag gtc          624                                                                          Leu Pro Leu Leu Ile Met Cys Ile Thr Tyr Ty - #r Arg Ile Phe Lys Val           #       205                                                                   - gcc cgg gat cag gcc aag agg atc aat cac at - #t agc tcc tgg aag gca          672                                                                          Ala Arg Asp Gln Ala Lys Arg Ile Asn His Il - #e Ser Ser Trp Lys Ala           #   220                                                                       - gcc acc atc agg gag cac aaa gcc aca gtg ac - #a ctg gcc gcc gtc atg          720                                                                          Ala Thr Ile Arg Glu His Lys Ala Thr Val Th - #r Leu Ala Ala Val Met           225                 2 - #30                 2 - #35                 2 -       #40                                                                           - ggg gcc ttc atc atc tgc tgg ttt ccc tac tt - #c acc gcg ttt gtg tac          768                                                                          Gly Ala Phe Ile Ile Cys Trp Phe Pro Tyr Ph - #e Thr Ala Phe Val Tyr           #               255                                                           - cgt ggg ctg aga ggg gat gat gcc atc aat ga - #g gtg tta gaa gcc atc          816                                                                          Arg Gly Leu Arg Gly Asp Asp Ala Ile Asn Gl - #u Val Leu Glu Ala Ile           #           270                                                               - gtt ctg tgg ctg ggc tat gcc aac tca gcc ct - #g aac ccc atc ctg tat          864                                                                          Val Leu Trp Leu Gly Tyr Ala Asn Ser Ala Le - #u Asn Pro Ile Leu Tyr           #       285                                                                   - gct gcg ctg aac aga gac ttc cgc acc ggg ta - #c caa cag ctc ttc tgc          912                                                                          Ala Ala Leu Asn Arg Asp Phe Arg Thr Gly Ty - #r Gln Gln Leu Phe Cys           #   300                                                                       - tgc agg ctg gcc aac cgc aac tcc cac aaa ac - #t tct ctg agg tcc aac          960                                                                          Cys Arg Leu Ala Asn Arg Asn Ser His Lys Th - #r Ser Leu Arg Ser Asn           305                 3 - #10                 3 - #15                 3 -       #20                                                                           - gcc tct cag ctg tcc agg acc caa agc cga ga - #a ccc agg caa cag gaa         1008                                                                          Ala Ser Gln Leu Ser Arg Thr Gln Ser Arg Gl - #u Pro Arg Gln Gln Glu           #               335                                                           - gag aaa ccc ctg aag ctc cag gtg tgg agt gg - #g aca gaa gtc acg gcc         1056                                                                          Glu Lys Pro Leu Lys Leu Gln Val Trp Ser Gl - #y Thr Glu Val Thr Ala           #           350                                                               #              1080ca gac agg taa                                             Pro Gln Gly Ala Thr Asp Arg                                                           355                                                                   - <210> SEQ ID NO 15                                                          <211> LENGTH: 359                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 15                                                          - Met Ala Pro Asn Gly Thr Ala Ser Ser Phe Cy - #s Leu Asp Ser Thr Ala         #                 15                                                          - Cys Lys Ile Thr Ile Thr Val Val Leu Ala Va - #l Leu Ile Leu Ile Thr         #             30                                                              - Val Ala Gly Asn Val Val Val Cys Leu Ala Va - #l Gly Leu Asn Arg Arg         #         45                                                                  - Leu Arg Asn Leu Thr Asn Cys Phe Ile Val Se - #r Leu Ala Ile Thr Asp         #     60                                                                      - Leu Leu Leu Gly Leu Leu Val Leu Pro Phe Se - #r Ala Ile Tyr Gln Leu         # 80                                                                          - Ser Cys Lys Trp Ser Phe Gly Lys Val Phe Cy - #s Asn Ile Tyr Thr Ser         #                 95                                                          - Leu Asp Val Met Leu Cys Thr Ala Ser Ile Le - #u Asn Leu Phe Met Ile         #           110                                                               - Ser Leu Asp Arg Tyr Cys Ala Val Met Asp Pr - #o Leu Arg Tyr Pro Val         #       125                                                                   - Leu Val Thr Pro Val Arg Val Ala Ile Ser Le - #u Val Ile Ile Trp Val         #   140                                                                       - Ile Ser Ile Thr Leu Ser Phe Leu Ser Ile Hi - #s Leu Gly Trp Asn Ser         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Arg Asn Glu Thr Ser Lys Gly Asn His Thr Th - #r Ser Lys Cys Lys Val         #               175                                                           - Gln Val Asn Glu Val Tyr Gly Leu Val Asp Gl - #y Leu Val Thr Phe Tyr         #           190                                                               - Leu Pro Leu Leu Ile Met Cys Ile Thr Tyr Ty - #r Arg Ile Phe Lys Val         #       205                                                                   - Ala Arg Asp Gln Ala Lys Arg Ile Asn His Il - #e Ser Ser Trp Lys Ala         #   220                                                                       - Ala Thr Ile Arg Glu His Lys Ala Thr Val Th - #r Leu Ala Ala Val Met         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Gly Ala Phe Ile Ile Cys Trp Phe Pro Tyr Ph - #e Thr Ala Phe Val Tyr         #               255                                                           - Arg Gly Leu Arg Gly Asp Asp Ala Ile Asn Gl - #u Val Leu Glu Ala Ile         #           270                                                               - Val Leu Trp Leu Gly Tyr Ala Asn Ser Ala Le - #u Asn Pro Ile Leu Tyr         #       285                                                                   - Ala Ala Leu Asn Arg Asp Phe Arg Thr Gly Ty - #r Gln Gln Leu Phe Cys         #   300                                                                       - Cys Arg Leu Ala Asn Arg Asn Ser His Lys Th - #r Ser Leu Arg Ser Asn         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Ala Ser Gln Leu Ser Arg Thr Gln Ser Arg Gl - #u Pro Arg Gln Gln Glu         #               335                                                           - Glu Lys Pro Leu Lys Leu Gln Val Trp Ser Gl - #y Thr Glu Val Thr Ala         #           350                                                               - Pro Gln Gly Ala Thr Asp Arg                                                         355                                                                   __________________________________________________________________________

We claim:
 1. An isolated nucleic acid comprising at least 413 contiguousnucleotides of SEQ ID NO:14, wherein the 413 contiguous nucleotidescontain one or more of the mutations selected from the group consistingof T to C at nucleotide 398, A to T at nucleotide 525, A to G atnucleotide 620, A to G at nucleotide 649, A to G at nucleotide 692, andG to A at nucleotide 802, wherein the nucleotide numberings are relativeto SEQ ID NO:14.
 2. The nucleic acid of claim 1, wherein the nucleicacid comprises SEQ ID NO:1.
 3. The nucleic acid of claim 1, wherein thenucleic acid comprises SEQ ID NO:2.
 4. An isolated polypeptide encodedby the nucleic acid of claim 1, wherein the polypeptide comprises anamino acid sequence encoded by at least 413 contiguous nucleotides ofSEQ ID NO:14.
 5. A nucleic acid vector comprising the nucleic acid ofclaim
 1. 6. A nucleic acid primer consisting of a nucleotide sequenceselected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,and SEQ ID NO:11.
 7. A method of amplifying or sequencing a nucleicacid, the method comprising hybridizing a target nucleic acid with thenucleic acid primer of claim 6, and extending the nucleic acid primerusing the target nucleic acid as a template, wherein the target nucleicacid is suspected of containing an allelic single nucleotidepolymorphism.
 8. A cell containing the nucleic acid vector of claim 5.